DOI: 10.1111/jpn.12193
ORIGINAL ARTICLE
In ovo carbohydrate supplementation modulates growth and immunity-related genes in broiler chickens S. K. Bhanja, A. Goel, N. Pandey, M. Mehra, S. Majumdar and A. B. Mandal Central Avian Research Institute, Izatnagar, UP, India
Summary A study was undertaken to investigate the role of in ovo administrated carbohydrates on the expression pattern of growth and immune-related genes. In ovo injections (n = 400) were carried out on the 14th day of incubation into the yolk sac/amnion of the broiler chicken embryos. Expression of growth-related genes: chicken growth hormone (cGH), insulin-like growth factor-I & II (IGF-I & II) and mucin were studied in hepatic and jejunum tissues of late-term embryo and early post-hatch chicks. Expression of candidate immune genes: Interleukin-2, 6, 10 and 12 (IL-2, IL-6, IL-10 and IL-12), Tumour necrosis factor-alpha (TNF-a) and Interferon gamma (IFN-c) were studied in peripheral blood monocyte cells of in ovo-injected and control birds following antigenic stimulation with sheep RBC (SRBC) or mitogen concanavalin A (Con-A). Glucose injection significantly increased the expression of IGF-II gene during embryonic period and both cGH and IGF-II in early post-hatch period, while ribose-injected chicks had higher expression of IGF-II gene during embryonic stage. Enhanced mucin gene expression was also observed in fructose-injected chicks during embryonic age. Glucose-injected chicks had higher expression of IL-6 or IL-10, while those injected with fructose or ribose had higher expression of IL-2, IL12 and IFN gamma. It is concluded that in ovo supplementation of carbohydrates might help in improving the growth of late-term embryos and chicks. In ovo glucose could modulate humoral-related immunity, while fructose or ribose might help in improving the cellular immunity in broiler chickens. Keywords in ovo injection, carbohydrates, gene expression, growth and immunity, broiler chickens Correspondence Dr. S. K. Bhanja, Principal Scientist, PHM Section, Central Avian Research Institute, Bareilly 243122, (Uttar Pradesh) India. Tel.: 91-9359105979; Fax: 91-5812301321; E-mail: [email protected] Received: 12 July 2013; accepted: 27 March 2014
During pre- and early post-hatch period, the chicks utilize their energy reserves to meet high demand for carbohydrates. As the amount of carbohydrates in the egg is very low, gluconeogenesis from protein is the source of glucose for accumulation of glycogen that eventually fuels the hatching activities (Klasing, 1998). When energy status is limited, hatchlings may lose weight and subsequently the development of critical tissues hampered. Moreover, in most of the commercial poultry operations, chicks get access to feed several hours after hatching. These limitations may be alleviated by administering refined carbohydrates or amino acids into the amnion or yolk sac at different days of incubation by a noble method called in ovo feeding, thus improving the chick’s liveability and growth (Bhanja et al., 2004b; Uni and Ferket, 2004). The in ovo fed nutrients help in accelerating enteric development in broilers and turkeys for greater
digestive and nutrient absorptive capacity during posthatch period (Uni and Ferket, 2004). It had already been reported that addition of glucose in drinking water suppressed gluconeogenic enzyme activity (Donaldson, 1995), thereby sparing the catabolism of useful proteins and antibodies. Studies conducted at our laboratory also revealed that glucose injection at later stage of embryonic development not only improved the broiler chick weight but also helped in the development of digestive tract (Bhanja et al., 2008a,b). Bhattacharyya et al. (2007) reported that in ovo injection of 1 ml, 10% glucose in turkey eggs on 25th day of incubation increased antisheep red blood cell (SRBC) titre (humoral immunity) and weight of bursa of fabricious at 28 days post-hatch, however, no affect was observed on cell-mediated immune response to PHAP (Phytohaemagglutinin-P). Earlier studies conducted on growth factors revealed that circulating IGF-1 and IGF-2 as well as hepatic IGF-1 mRNA expressions were regulated by
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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Introduction
Carbohydrates and gene expressions
S. K. Bhanja et al.
nutritional state (Thissen et al., 1994). In avian species, expression of IGF-1 was regulated in a growth hormone (GH) dependent manner, during post-natal development (McMurtry et al., 1997). In spite of growth function, nutrients also play an important role in the functioning of the immune system. Upon activation, naive T helper cells (Th0) differentiate into either T helper 1 (Th1) or Th2 effector cells and secrete different cytokines that mediate immune responses (Spellberg and Edwards, 2001). Th1 type responses are primarily cell-mediated immunity and inflammation, while Th2 type are cytokines mediating humoral immunity. It will be interesting to see whether the nutrients could be able to modulate expression of certain cytokines, thus mediating humoral or cellular immunity. Scanty information is available on the role of critical macro/micro-nutrients on the expression of growth and immunity-related genes in chickens. Hence, keeping in view of the above facts, an effort was made to assess the effect of in ovo supplementation of carbohydrates (glucose, fructose or ribose) on the expression profile of growth and immune-related genes in broiler chickens during late embryonic and early post-hatch periods. Such understanding will help in formulating diets of neonatal chicks for better growth and immune responsiveness.
of the embryo, under laminar flow system, following standard procedure of Bhanja et al. (2004a). Briefly, first the target site (yolk sac/amnion) in the egg was standardized by injecting 0.5 ml solution of Indian ink through pinhole created at the broad end of a 14thday embryonated egg using different length of needles. Immediately after the injection, the shell of the respective end was cut with dental drill, and egg shell and shell membranes were removed with forceps. The injection site was visually observed. Based on the deposition site, the needle which reached to yolk sac/ amnion of 14th-day embryonated egg maximum time was selected for in ovo supplementation of nutrients. Before in ovo injection, the broad end of the egg was sterilized with 70% alcohol and a pin hole was made. With the help of syringe containing 24 gauge 25 mm hypodermic needle 0.5 ml sterile water containing carbohydrate (50 mg each) was injected into the yolk sac/amnion of embryo. After in ovo injection, the site was sealed with sterile paraffin and eggs were returned to the incubator. On the 19th day of incubation, the eggs were placed in pedigree-hatching boxes. Post-hatch, the chicks were reared in electrically operated battery brooders and provided standard nutrition and management in accordance with Institutional Ethics Committee at Central Avian Research Institute, Izatnagar, India till 42nd day of age.
Materials and methods
Collection of tissues for growth-related genes
Experimental birds and treatments
Four embryos/chicks from each group were killed at different ages (18th and 20th ED, day old, 7th, 10th and 14th day post-hatch). About 200 mg of liver (for cGH, IGF-I IGF-II gene expression) and intestinal tissue (jejunum, for mucin gene expression) from each bird were collected. The tissue was then homogenized using automated homogenizer (Polytron) to disrupt the cells and to remove the debris. 50 mg of homogenized tissue was used for total RNA isolation and the rest, 150 mg of tissue, was immersed in 500 ll RNA stabilizing solution and kept at 4 °C overnight and then transferred to 80 °C deep freeze for contingency uses.
The study was conducted using carbohydrates (glucose, fructose and ribose) as in ovo nutrients at a concentration of 50 mg/egg. 400 fertile eggs collected from coloured synthetic broiler breeder flocks (Cross of Cornish and Plymouth Rock) maintained on balanced ration were distributed into four groups (threetreatments and one un-injected control) of 100 eggs each. In our earlier studies on in ovo feeding (Bakyaraj et al., 2012), no statistical difference was observed with respect to percent hatchability and post-hatch performance of un-injected control group and sham control (only placebo no nutrients) group; hence, sham control group was excluded from the present experimental design. After collection, the eggs were fumigated (at 19 concentration) immediately and incubated in a forced draft incubator at a temperature of 37.5 °C with relative humidity of 60%.
Differential expression of candidate immune function genes
In ovo administration of nutrients were carried out on the 14th day of incubation into the yolk sac/amnion
To quantify the candidate genes of humoral immunity like IL-6, IL-10 and TNF-a, four birds from each group were injected intravenously with 1.0 ml, 1% (V/V) sheep red blood cells (SRBC) on 14th day post-hatch and 5 days post-injection, blood was collected from jugular vein. Approximately 1.5 ml blood from each
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In ovo feeding
S. K. Bhanja et al.
bird was mixed with 3.0 ml of PBS (1:2). For separation of peripheral blood mononuclear cells (PBMC), 3.0 ml Histopaque-1.077 (Sigma Diagnostics, St. Louis, MO, USA) was taken in a 15.0 ml conical centrifuge tube, and the diluted blood was carefully layered over the histopaque and centrifuged at 471 g for exactly 20 min at room temperature. After centrifugation, the opaque interface containing PBMC was carefully transferred to a clean conical centrifuge tube and 10.0 ml isotonic PBS solution was added and mixed by gentle aspiration then centrifuged at 910 g for 10 min. This step was repeated twice to ensure complete washing. After centrifugation, pellet was dissolved in denaturing solution for total RNA isolation. For quantification of cellular immune-related genes like IL-2, IL-12 and IFN gamma, approximately, 1.5 ml blood was collected from the jugular vein of four birds per group on the 21st day post-hatch and PBMC cells were separated (as detailed earlier). Viability of these cells were determined by Trypan blue (0.4%) staining method. Cells were re-suspended in known volume of RPMI-1640 (Sigma) to make the final concentration of 106 cells per ml of medium. PBMCs were then plated in 6-well culture plates with RPMI-1640 (without phenol red) medium, supplemented with 10% FBS, 2 mM L-glutamine, 2 mM L-argenine under 5% CO2 tension in humidified atmosphere. The PBMC cells were sensitized to 10 lg/ml concanavalin-A (Con-A) for a period of 4 h; then the PBMCs adhered to culture plates were harvested in to a 1.5 ml eppendorf tube and centrifuged at 3640 g for 1 min. After centrifugation, the pellet was dissolved in denaturing solution for total RNA isolation.
Carbohydrates and gene expressions
First strand cDNA synthesis by RT-PCR
Concentration of each RNA sample was computed and then adjusted with RNA free water to make a uniform concentration (2000 ng/ll). Required amount of RNA from each sample was taken as template and first strand cDNA was prepared by Revert AidTM first strand cDNA synthesis kit (MBI, Fermentas Inc., Hanover, MD, USA). This first stand cDNA was used as template for amplification of different genes by polymerase chain reaction (PCR) or stored at ultra-low temperature for further use. Standardization of primers for PCR
Oligonucleotide primers (Table 1) were designed, synthesized and standardized for PCR reaction conditions. PCR products of different genes were sequenced for nucleotide bases and the sequences were submitted to NCBI, USA or EMBL, UK for confirmation, and accession numbers were obtained for those genes . Polymerase chain reaction
The PCR amplification was carried out in 25 ll volume, containing 10 pM of each primer, 0.1 mM dNTP mix, 1 unit of Taq DNA polymerase (Fermentas) and 1 ll cDNA in 19 Taq polymerase buffer. Cycling conditions were: initial denaturation at 94 °C (5 min); 35 cycles of denaturation at 94 °C (45s), annealing temperature (as given in Table 1) for 1 min and extension at 72 °C (1 min); and final extension at 72 °C (10 min). Semi-quantitative analysis
Total RNA isolation
The samples collected for expression of growth and immunity-related genes were subjected to total RNA isolation (RNAgents, Promegaâ, Madison, WI, USA) as per the manufacture instructions. The concentrations and purities of RNA preparations were determined spectrophotometrically by taking absorbance at 260 and 280 nm in a Nanodrop instrument. It was ensured that A260/A280 ratio of the samples was >1.8. The integrity of RNA was also assessed by UV visualization of intact 28S and 18S bands on a 2% agarose gel. The possible trace of genomic DNA was removed by treating 5 lg of RNA samples with 5 U of RNase-free DNase at 37 °C for 1 h. The DNase was subsequently inactivated by incubating at 65 °C for 10 min. The RNA samples were stored at –80 °C till further processing. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
Electrophoresis was performed on 2% Agarose having Ethedium Bromide stain at 2 V/cm for 2 to 3 h. The gel was then examined under UV light and photographed in a Gel documentation system (Bio-Rad). Image J analysis software was used for quantification of luminance (integrated density). Housekeeping (28S rRNA) gene expression of respective sample was used for normalization of target gene expression. Ratio of gene expression for each sample was calculated using the formula: Ratio expression integrated density of target gene ¼ Integrated density of housekeeping gene (28S rRNA) The average ratio of un-injected control group was used to calculate the fold change in target 3
4
Interleukin-6 (IL-6)
Interleukin-10 (IL-10)
Tumour Necrosis Factor –alfa (TNFa)
Interleukin-2 (IL-2)
Interleukin-12 (IL-12)
5
6
7
8
9
28S r RNA (Reference)
Mucin gene
4
11
Insulin-like growth factor-II (IGF-II)
3
Interferon gamma (IFNc)
Insulin-like growth factor-I (IGF-I)
2
10
Chicken Growth hormone gene (cGH)
Gene
1
Sl No. F-caccacagctagagacccacatc R-cccaccggctcaaactgc F-ggtgctgagctggttgatgc R-cgtacagagcgtgcagatttaggt F-ggcggcaggcaccatca R-cccggcagcaaaaagttcaag F-ctggctccttgtggctcctc R-agctgcatgactggagacaactg F-gaaatccctcctcgccaatctga R-tgaaacggaacaacactgccatct F-ctgaaggcgacgatgc R-ttcctcctcctcatcagc F-agaccagatgggaagggaatgaa R-gaagaggccaccacacgacag F-cccgtggctaactaatctgctg R-tgagacaccagtgggaaacagt F-gccgactgagatgttcctgg R-ccttgcttttgtatttctttgtgc F-agctgacggtggacctattattgt R-cggctttgcgctggattc F-caggtgcagatcttggtggtagta R- gctcccgctggcttctcc
Primer (5I–3I)
Table 1 Oligonucleotide sequence of growth and immune-related gene primers
58
58
57
57
55
57
55
58
58
58
58
Annealing Temp. (oC)
273
260
227
287
219
179
219
242
215
203
201
Size of Amplicon (bp)
House keeping
Cell mediate immunity
Cell mediate immunity
Cell mediate immunity
Humoral Immunity
Humoral Immunity
Humoral Immunity
Intestinal tract Dev.
Growth
Growth
Growth
Putative biological role
JN639848
JN942588
JN942590
HE608819
JN942589
EF554720
JN639847
JN639849
JN942579
JN942578
HE608816
Accession number
Carbohydrates and gene expressions S. K. Bhanja et al.
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
S. K. Bhanja et al.
Carbohydrates and gene expressions
gene expression of treated samples using the formula: Fold Change (each sample) Ratio (each sample) ¼ Average ratio of un-injected control group The resulted fold change in gene expression of each treatment group was used for statistical analysis. As the average ratio of un-injected control group was used to find the fold change in the treatment groups, so the fold change in un-injected control group was always one.
afterwards, 100 lg PHA-P, dissolved in 0.1 ml PBS, was injected into the same interdigital space of the right foot. The toe web of the left foot was used as control and injected only with PBS. The inflammatory response was determined 24 h later by measuring the thickness of the respective toe-webs and subtracting the earlier measurements by using formula (Corrier and Deloach, 1990), Foot Web Index = (R2–R1)–(L2– L1) where R2 – thickness after 24 h of PHA-P injection, R1 – thickness before injection of PHA-P injection, L2 – thickness after 24 h of PBS injection and L1 – thickness before injection of PBS injection. Statistical analysis
In-vivo immune responses
Sheep red blood cell suspended in Alsever’s solution were washed three times in isotonic phosphatebuffered saline (PBS: pH 7.2) using centrifugation (328 g) and adjusted to provide a 1% suspension (v/v) which was stored at 4 °C prior to use. Ten chicks from each group (3-week-old) were injected intravenously with 1 ml of the SRBC suspension. Five days later, a blood sample (2.0 ml) was obtained from the jugular vein of each chick. Each blood sample was allowed to clot for serum collection and sera were stored at 20 °C until analysis. The antibody response to SRBC was determined using a standard haemagglutination assay (Siegel and Gross, 1980). The reciprocal of highest dilution showing clear agglutination was the end point of titre and the values were expressed as log 2. The cellular immune response was also assessed in another ten chicks (3-week-old) using the in-vivo cutaneous basophilic hypersensitivity response to the lectin phytohaemagglutinin from Phaseolus vulgaris (PHA-P). The toe web thicknesses, between the third and fourth digits, of both left and right feet were measured using a micrometre. Immediately
Fold changes in expression of growth-related genes for a particular age was analysed by one-way ANOVA, whereas the multivariate analysis (taking treatment and age as factors) was done to see treatment/period effect using SPSS software package Ver 16.0 (2007). Difference in mean values was considered as significant at the level of 95% (p < 0.05) and 99% (p < 0.01) using Duncan’s multiple range test (Duncan, 1955). The growth response data and immunological parameters were analysed by one-way ANOVA only. Results Expression of growth-related genes
Gel photographs showing integrated densities of target gene and a housekeeping gene (28s ribosomal RNA) for a particular sample during pre- and post-hatch period have been presented in Figures 1 and 2. Variation in the intensities of target and housekeeping gene was noticed within and between groups; the ratio of relative intensity was calculated to derive the fold expression as described in the earlier section and are presented in Table 2 and Figure 3.
(a)
(b) (c)
Fig. 1 Gel photographs showing band intensity (four per treatment) of growth-related genes and a house-keeping gene (28s RNA) during embryonic stages in in ovo glucose- (G), fructose- (F) or ribose (R)-injected and un-injected control (C) embryos. (a) Glucose showing higher hepatic cGH and ribose showing higher IGF-II gene expression on 18th day of embryonic age (b) Fructose showing higher jejunal mucin gene expression on 18th day of embryonic age (c) Fructose and ribose showing higher hepatic cGH gene expression on 20th day of embryonic age.
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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S. K. Bhanja et al.
Carbohydrates and gene expressions
(a)
(b) (c) (d) Fig. 2 Gel photographs showing band intensity (four per treatment) of growth-related genes and a house-keeping gene (28s RNA) during post-hatch periods in in ovo glucose- (G), fructose- (F) or ribose (R)-injected and un-injected control (C) chicks. (a) Glucose showing higher hepatic cGH, IGF-I and IGF-II gene expressions on 7th day post-hatch age (b) Glucose showing higher hepatic cGH gene expression on 10th day post-hatch age (c) Glucose showing higher hepatic IGF-I gene expressions on 14th-day post-hatch age (d) Ribose showing higher jejunal mucin gene expressions on 7th-day posthatch age.
Pre-hatch period
When data was analysed by two way ANOVA to see period and treatment interaction, irrespective of period of study higher hepatic cGH (1.43–1.58-fold) and IGF-I (1.15–1.28) expression but not differing (p > 0.05) from control was observed in all the carbohydrate-injected embryos. Hepatic cGH expression was apparently higher in glucose (18th ED) or in fructose or ribose-injected embryos (20th ED) than un-injected embryos (Figure 3). Whereas IGF-II expression was higher (p < 0.05) in glucose and ribose-injected embryos compared with control embryos, the fructose group had intermediate values. Higher mucin gene expression (p < 0.01) was observed in fructose-injected embryos. When expression was compared for different embryonic ages,
higher hepatic cGH at 18th or 20th ED, IGF-II and mucin gene expression at 18th ED was observed than those at DOH (Table 2). There was no significant interaction between period and treatment in cGH or IGF-I expression. However, interaction was significant for IGF-II and mucin gene where glucoseor ribose-injected embryos had higher IGF-II expression and fructose-injected embryos had higher mucin gene expression at 18th ED (Figure 3). Post-hatch period
When expression was compared for different posthatch (PH) periods cGH and mucin gene expression was higher on 7th and 10th PH, while IGF-I and IGFII expression was higher only on 7th PH. Between treatments cGH and IGF-II gene expression was
Table 2 Mean fold change in the expression pattern of growth-related genes in hepatic and jejunal tissues of four embryos/chicks in different carbohydrate treatments at pre- and post-hatch period Embryonic
Age 18th ED 20th ED DOH Treatments Glucose Fructose Ribose Control SEm Significance (Probability) Period Treatment Period 9 Treatment
Post-hatch
cGH
IGF-I
IGF-II
Mucin
1.46b 1.71b 0.95a
1.29 1.03 1.12
3.05b 1.16a 0.94a
1.43b ND 1.04a
1.43 1.49 1.58 1.00 0.107
1.16 1.28 1.15 1.00 0.061
2.11b 1.64ab 2.12b 1.00a 0.234
1.07a 1.71b 1.16a 1.00a 0.097
0.01 NS NS
NS NS NS
0.01 0.05 0.01
0.01 0.01 0.01
cGH
IGF-I
IGF-II
Mucin
7th PH 10th PH 14th PH
1.67c 1.27b 0.95a
1.38b 0.99a 0.90a
1.23b 1.03a 1.11ab
1.22b 1.01b 0.68a
Glucose Fructose Ribose Control SEm
1.66b 1.22a 1.31a 1.00a 0.081
1.22 1.18 0.97 1.00 0.062
1.36b 1.12a 1.01a 1.00a 0.038
0.73a 0.94ab 1.21b 1.00ab 0.063
0.01 0.01 0.01
0.01 NS 0.01
0.01 0.01 0.05
0.01 0.01 NS
Values are mean of four observations. Means bearing different superscripts in a column differ significantly (p < 0.05).
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Carbohydrates and gene expressions
(a)
(b)
(c)
(d)
Fig. 3 Mean fold increase in the expression pattern of growth-related genes during embryonic and post-hatch period in glucose-, fructose- or riboseinjected embryos compared with un-injected control embryos. (a) Hepatic chicken growth hormone (b) Hepatic insulin-like growth factor-I (c) Hepatic insulin-like growth factor-II (d) Jejunal mucin gene.
significantly higher in glucose-injected chicks. No variation was observed in the expression of IGF-I gene; however, the expression of mucin gene was significantly higher in ribose-injected chicks than glucoseinjected chicks (Table 2). Significant interaction between period and treatment was observed for cGH, IGF-I and IGF-II gene, where in ovo glucose-injected chicks had higher expression of hepatic cGH on 7th and 10th PH, IGF-I gene on 7th PH and IGF-II expression on 7th and 14th day PH than control chicks (Figure 3). Expression of immune-related genes
The relative expression of humoral immunityrelated genes (IL-6, IL-10 and TNF alpha) have been presented in Figure 5. Glucose- or fructoseinjected chicks had significantly higher expression (p < 0.01) of IL-6 gene compared with control, but down-regulated expression was observed in ribosetreated birds. Expression of IL-10 was higher (p < 0.01) in glucose-injected chicks, but the expression decreased in fructose- or ribose-injected chick compared to control. There was no variation in the expression of TNF alpha gene between glucose or fructose treatment and control, but, ribosetreated chicks showed significant down-regulation (p < 0.01) of TNF alpha gene. The relative expression of cellular immunity cytokines, IL-2, IL-12 and IFN gamma at 14th day
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
post-hatch have been presented in Figure 4 and 5. Expression of IL-2 gene was significantly higher (p < 0.01) in fructose- or ribose-injected chicks compared with un-injected control; however, the expression in glucose-injected chicks was down-regulated in comparison to fructose- or ribose-injected chicks. Expression of IL-12 gene was significantly higher (p < 0.05) in ribose-treated chicks than those of glucose, but no difference was observed between carbohydrate-treated chicks and control. Expression of IFN gamma was significantly (p < 0.01) higher in ribose- or fructose-injected birds compared with control, but down-regulated in glucose-injected chicks. In-vivo immune response to SRBC and PHA-P
Humoral immunity (response to SRBC antigen measured as HA titre, Log2 value) was higher (p < 0.05) in ribose-injected chicks than control; however, titre
Fig. 4 Gel photographs showing band intensity of Cell-Mediated Immune genes and a house-keeping gene (28s RNA) at 21 d post-hatch in in ovo glucose- (G), fructose- (F) or ribose (R)-injected and un-injected control (C) chicks after sensitizing the PBMC cells with 10 lg Con-A/mL of RPMI-1640 media.
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Carbohydrates and gene expressions
(a)
(b)
Fig. 5 Differential expression of immunity-related genes showing mean fold change over control group in in ovo carbohydrates-injected chicks (a) Glucose- or fructose-treated chicks showing higher expression of humoral immune-related genes in PBMC following SRBC challenge (b) Fructoseor ribose-treated chicks showing higher expression of cellular immune genes in PBMC after sensitizing with Con-A.
Table 3 Effect of in ovo injection of carbohydrates on the antibody titre (response to SRBC injection) and toe web thickness (response to PHAP) of ten 3-week-old broiler chickens
In the present study, in ovo glucose injection resulted in significantly higher expression of cGH gene during post-hatch period. Bhanja et al. (2008b) reported that in ovo injection of glucose on 14th day of incubation did not affect the hatchability and chick weight but had higher levels of plasma glucose and plasma protein on the DOH. In a recent study, Zhai et al. (2011a, b,c) reported that embryo weight relative to set egg weight on 19th day of incubation increased by injection of carbohydrates (glucose, sucrose, maltose and dextrin). These findings corroborate our result, as cGH
expression was on peak at 20th day of incubation. Moreover, studies conducted at our laboratory on in ovo feeding of amino acids and vitamins had significantly higher cGH expression (Bhanja, S.K., personal communication), Thus suggesting the role of in ovo feeding of nutrients for enhancing overall growth and proliferation of tissues in neonatal chicks. IGF-I levels increased steadily during incubation from day 6 to peak at day 15 of incubation but reduced during hatch (Robcis et al., 1991). In the present study also, the expression of hepatic IGF-1 gene was apparently higher on the 18th day of incubation and showed a decreasing trend towards hatch. During post-hatch period, higher IGF-1 expression was observed on 7th than 10th or 14th PH in glucose-injected chicks. Contrary to this Lu et al. (2007) reported that after hatch, IGF-1 levels rapidly increased and kept increasing all the way until 21 days post-hatch. Deprem and G€ ulmez (2007) reported that in ovo rhIGF-1 injection in quails was found to accelerate skeletal muscle development in quail embryos. The expression of cGH gene was higher at 18th and 20th ED but no variation was observed in the expression of IGF-I gene during this period suggesting IGF-I gene expression is GHindependent during embryonic stage as that of reported earlier (Tanaka et al., 1996) but acts as a mediator of many of the actions of GH and stimulator of tissue growth and differentiation in post-hatch period (Cohick and Clemmons, 1993). In this study also, during post-hatch period the expression of cGH and IGF-1 gene followed a similar trend and in ovo glucose injection had higher expression of both the genes although not significant for IGF-I. Foye (2005) reported that in poultry as in mammalian species, IGF-I and II stimulate hepatic glycogen, RNA and protein synthesis. The expression of IGF-II gene during incubation was found to be highest at 18th ED; however, during PH, the expression had
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Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
Treatments
Anti-SRBC Titre (log 2)
Foot web Index (mm) in response to mitogen PHAP
Glucose Fructose Ribose Control Pooled SEM Sig. Level
9.71ab 9.71ab 10.83b 9.00a 0.22 p = 0.022
0.3500a 0.4650ab 0.6575b 0.4729ab 0.040 p = 0.039
Values are mean of ten observations. Means bearing different superscripts in a column differ significantly (p < 0.05).
value in glucose or fructose-injected chicks were not different from ribose or control chicks (Table 3). Cellular immunity (foot web index, response to PHA-P injection) was similar among carbohydrate-injected and un-injected control chicks, although response was better in ribose-injected chicks (Table 3) but it did not reach to a significant level (p > 0.05). Discussion Growth-related genes
S. K. Bhanja et al.
shown decreasing trend with increase in age. This correlates the earlier findings where IGF-II is reported as an important functionary for chick embryonic development (McMurtry et al., 1998) and plasma IGF-II level decreased from 60 ng/ml on 17th of incubation to 25 ng/ml at 21 day post-hatch (Lu et al., 2007). Fructose-injected embryos had higher expression of jejunal mucin gene at 18 ED, whereas, ribose-injected chick had significantly higher expression on 7th day post-hatch than glucose-injected chicks. Uni and Ferket (2004), reported that in ovo feeding of carbohydrates at 18 day of incubation resulted significant increase in jejunum villi height by over 45% even 48 h after injection. Higher mucin gene expression and goblet cell density in jejunum villi was reported in in ovo carbohydrate-injected embryos/chicks than the saline-control group (Smirnov et al., 2006). Bhanja et al. (2008a) had also reported better gastrointestinal tract development in glucose-injected chicks. In another study, in ovo mannan oligosaccharide feeding at 17th or 18th day of incubation, resulted in the 3 -fold increase of mucin 2 gene expression (CheledShoval et al., 2011). These studies suggested that the presence of carbohydrate in the intestinal lumen of the chicken embryo might have triggered the goblet cells for production of acidic mucin. From our study, the role of fructose in enteric development cannot be ruled out as it increased the mucin gene expression during pre-hatch period.
Carbohydrates and gene expressions
Development of both B and T lymphocytes is initiated during embryogenesis and continues till post-hatch. These cells play an important role in immunity as native T cells differentiate into Th1 cells (cellular immunity) and Th2 cells (humoral immunity). Glick et al. (1981, 1983), reported that the thymus is very sensitive to periods of food deprivation, which caused a rapid decline in CD4+ T cells resulting in lower IgG production. The CD4+ cells act as the source for IL-6, a pro-inflammatory cytokines, which induces the final maturation of B cells into antibody-secreting plasma cells (Jones, 2005) causing the proliferation and differentiation of immunoglobins. In the present study, expression of humoral immune-related genes, that is, IL-6 and IL-10, were increased in glucose treatment group in comparison to un-injected control. This is in line with the earlier study of Greiner et al. (1994), who reported that glucose is an essential fuel for proliferating Th2 cells for the production of antibody, thus providing humoral immunity. Studies in human
diabetic individuals revealed that hyperglycaemia acutely increased circulating cytokine concentrations of IL-6 and TNF-a by an oxidative mechanism and played a role in the immune activation in diabetes (Esposito et al., 2002). In another study, Humphrey and Rudrappa (2008) reported that glucose availability induced metabolic changes in thymocytes that altered their energy status and influence the development and differentiation of T cells in the chicken thymus (Zhang et al., 2005). They had also reported that thymocytes developing in low glucose concentrations (5 mM) had elevated rates of apoptosis. Therefore, increasing the in ovo availability of glucose might have increased the synthesis of trophic factors which promote T-cell glucose metabolism and helps in the development of the chick’s immune system at early life. In-vivo response to SRBC antigen was significantly higher in ribose-injected birds than un-injected control but not different from that of glucose- or fructoseinjected chicks. However, Bhattacharyya et al. (2007) reported that in ovo injection of 1 ml of 10% glucose to turkey eggs increased anti-SRBC titre (humoral immunity) and weight of bursa of fabricious. It is reported that Th1 and Th2 responses are also cross-regulated, wherein IFN-gamma, a cytokine produced by Th1 cells, inhibits IL-4 production and suppresses Th2 development (Gajewski and Fitch, 1988). Conversely, IL-4 and IL-10 produced by Th2 cells block differentiation of Th0 to Th1 (D’Andrea et al., 1993). In the present study, expression of IL-10 was higher in glucose-injected chicks, but the expression decreased in fructose or ribose-injected chick compared to control chicks. It was also interesting to note that the expression of IL-2 and IFN gamma gene was significantly higher in fructose or ribose treated chicks but no difference was observed in glucose treatment as compared to control. Earlier studies on critical amino acids had demonstrated that either in ovo feeding (Bhanja et al., 2012) or dietary supplementation (Sijben et al., 2001) increases the PHA-P response and expression of cellular immune genes (IL-2 and IL-12) in broiler chicks. Similarly, Bhanja and Mandal (2005) also found a significant difference in cell-mediated immunity in in ovo injection of amino acid. The present study on cellular immune response to mitogen PHA-P revealed no significant difference between carbohydrates-injected and control chicks, but apparently higher response was observed in ribose-injected chicks. In another study, Kadam et al. (2008) reported that in ovo injection of threonine in broiler eggs had no affect on cell-mediated immune response to PHA-P.
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Immunity-related genes
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Carbohydrates and gene expressions
In summary, the present investigation on in ovo feeding of carbohydrates revealed that glucose played an important role during late embryonic and early post-hatch period by increasing the expression of cGH and IGF-II genes. It also increased expression of humoral genes, IL-6 and IL-10. The role of in ovo supplemented fructose and ribose could not be ignored as their presence during late embryonic stage resulted in better expression of IGF-II or mucin gene. Additionally, ribose modulated the expression of cellular References Bakyaraj, S.; Bhanja, S. K.; Majumdar, S.; Dash, B. B., 2012: Post-hatch immunomodulation through in ovo supplemented nutrients in broiler chickens. Journal of the Science of Food and Agriculture 92, 313–320. Bhanja, S. K.; Mandal, A. B., 2005: Effect of in ovo injection of critical amino acids on pre and post hatch growth, immunocompetence and development of digestive organs in broiler chickens. AsianAustralasian Journal of Animal Science 18, 524–531. Bhanja, S. K.; Mandal, A. B.; Johri, T. S., 2004a: Standardization of injection site, needle length, embryonic age and concentration of amino acids for in ovo injection in broiler breeder eggs. Indian Journal of Poultry Science 39, 105–111. Bhanja, S. K.; Mandal, A. B.; Goswami, T. K., 2004b: Effect of in ovo injection of amino acids on growth, immune response, development of digestive organs and carcass yield of broiler. Indian Journal of Poultry Science 39, 212– 218. Bhanja, S. K.; Mandal, A. B.; Agarwal, S. K.; Majumdar, S.; Bhattacharyya, A.; Kadam, M., 2008a: In ovo glucose injection for higher chick weight and gastrointestinal tract development. The Indian Veterinary Journal 85, 289–292. Bhanja, S. K.; Mandal, A. B.; Agarwal, S. K.; Majumdar, S., 2008b: Effect of in ovo glucose injection on the post hatchgrowth, digestive organ development and blood biochemical profiles in broiler chickens. Indian Journal of Animal Science 78, 869–872. Bhanja, S. K.; Mandal, A.; Agarwal, S. K.; Majumdar, S., 2012: Modulation of post hatch-growth and immunocompetence through in ovo injection of limiting amino acids in broiler chickens. Indian Journal of Animal Science 92, 993–998.
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immune genes, IL-2, IFN gamma and IL-12 in broiler chickens. Acknowledgement The financial assistance provided by Department of Biotechnology, Ministry of Science and Technology, Govt. of India for carrying out this research work is duly acknowledged.
Bhattacharyya, A.; Majumdar, S.; Bhanja, S. K.; Mandal, A. B.; Dash, B. B.; Agarwal, S. K., 2007: In ovo injection of glucose supports immunecompetence. World Poultry–Turkey Special 10-11. Cheled-Shoval, S. L.; Amit-Romach, E.; Barbakov, M.; Uni, Z., 2011: The effect of in ovo administration of mannan oligosaccharide on small intestine development during the pre- and posthatch periods in chickens. Poultry Science 90, 2301–2310. Cohick, W. S.; Clemmons, D. R., 1993: The insulin-like growth factors. Annual Review of Physiology 55, 131–153. Corrier, D. E.; Deloach, J. R., 1990: Evaluation of cell mediated, cutaneous basophil hypersensitivity in young chickens by inter digital skin test. Poultry Science 69, 403–408. D’Andrea, A.; Aste-Amezaga, M.; Valiante, N. M.; Ma, X.; Kubin, M.; Trinchieri, G., 1993: Interleukin 10 (IL-10) inhibits human lymphocyte interferon-gamma production by suppressing natural killer cell stimulatory factor/ IL-12 synthesis in accessory cells. Journal of Experimental Medicine 178, 1041–1048. Deprem, T.; G€ ulmez, N., 2007: The effects of in ovo insulin-like growth factor-1 on embryonic development of musculus longus colli dorsalis in japanese quail. Turkish Journal of Veterinary and Animal Science 31, 233–240. Donaldson, W. E., 1995: Carbohydrate, hatchery stressors affect poult survival. Feedstuffs 67, 16–17. Duncan, D. B., 1955: Multiple range and multiple F tests. Biometrics 11, 1–42. Esposito, K. F.; Nappo, R.; Marfella, G.; Giugliano, F.; Giugliano, M. C.; Quagliaro, L.; Ceriello, A.; Giugliano, D., 2002: Inflammatory cytokine concentrations are acutely increased by hyperglycemia in humans: role of oxidative stress. Circulation 106, 2067–2072.
Foye, O. T., 2005: ‘The biochemical and molecular effects of amnionic nutrient administration, “in ovo feeding” on intestinal development and function and carbohydrate metabolism in turkey embryos and poults’, Dissertation Ph.D, North Carolina State University, Raleigh, NC. Gajewski, T. F.; Fitch, F. W., 1988: Antiproliferative effect of IFN gamma in immune regulation. I. IFN-gamma inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones. Journal of Immunology 140, 4245–4252. Glick, B.; Day, E. J.; Thompson, D., 1981: Calorie-protein deficiencies and the immune response of the chicken I. Humoral immunity. Poultry Science 60, 2494–2500. Glick, B.; Taylor, R. L. Jr; Martin, D. E.; Watabe, M.; Day, E. J.; Thompson, D., 1983: Calorie-protein deficiencies and the immune response of the chicken II. Cell-mediated immunity. Poultry Science 62, 1889–1893. Greiner, E. F.; Guppy, M.; Brand, K., 1994: Glucose is essential for proliferation and the glycolytic enzyme induction that provokes a transition to glycolytic energy production. Journal of Biological Chemistry 269, 31484–31490. Humphrey, B. D.; Rudrappa, S. G., 2008: Increased glucose availability activates chicken thymocyte metabolism and survival. Journal of Nutrition 138, 1153–1157. Jones, S. A., 2005: Directing transition from innate to acquired immunity: defining a role for IL-6. Journal of Immunology 175, 3463–3468. Kadam, M. M.; Bhanja, S. K.; Mandal, A. B.; Thakur, R.; Vasan, P.; Bhattacharyya, A.; Tyagi, J. S., 2008: Role of in ovo threonine supplementation on early growth, immunocompetence and digestive enzyme activity in broiler chickens. British Poultry Science 49, 736–741.
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Klasing, K. C., 1998: Carbohydrates. In Comparative Avian Nutrition. CAB Intlernational, New York, pp. 201–209. Lu, J. W.; McMurtry, J. P.; Coon, C. N., 2007: Developmental changes of plasma insulin, glucagon, insulin-like growth factors, thyroid hormones, and glucose concentrations in chick embryos and hatched chicks. Poultry Science 86, 673– 683. McMurtry, J. P.; Francis, G. L.; Upton, Z., 1997: Insulin-like growth factors in poultry. Domestic Animal Endocrinology 14, 199–229. McMurtry, J. P.; Rosebrough, R. W.; Brocht, D. M.; Francis, G. L.; Upton, Z.; Phelps, P., 1998: Assessment of developmental changes in chicken and turkey insulin-like growth factor-II by homologous radioimmunoassay. Journal of Endocrinology 157, 463–473. Robcis, H. L.; Caldes, T.; De Pablo, F., 1991: Insulin-like growth factor-I serum levels show a midembryogenesis peak in chicken that is absent in growthretarded embryos cultured ex ovo. Endocrinology 128, 1895–1901. Siegel, P. B.; Gross, W. B., 1980: Production and persistency of antibodies in chickens to sheep erythrocytes
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I. Directional selection. Poultry Science 59, 1–5. Sijben, J. W. C.; Schrama, J. W.; Parmentier, H. K.; Van der Poel, J. J.; Klasing, K. C., 2001: Effects of dietary polyunsaturated fatty acids on in vivo splenic cytokine mRNA expression in layer chicks immunized with salmonella typhimurium lipopolysaccharide. Poultry Science 80, 1164–1170. Smirnov, A.; Tako, E.; Ferket, P. R.; Uni, Z., 2006: Mucin gene expression and mucin content in the chicken intestinal goblet cells are affected by in ovo feeding of carbohydrates. Poultry Science 85, 669– 673. Spellberg, B.; Edwards, J. E. Jr, 2001: Type 1/Type 2 immunity in infectious diseases. Clinical Infectious Diseases 32, 76– 102. SPSS 2007: SPSS for Windows, Version 16.0. Released 2007. SPSS, Chicago. Tanaka, M.; Hayashida, Y.; Sakaguchi, K.; Ohkubo, T.; Wakita, M.; Hoshino, S.; Nakashima, K., 1996: Growth hormone independent expression of insulin-like growth factor I. messenger ribonucleic acid in extra hepatic tissues of the chicken. Endocrinology 137, 30–34.
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Thissen, J. P.; Ketelslegers, J. M.; Underwood, L. E., 1994: Nutritional regulation of insulin-like growth factors. Endocrinology Review 15, 80–101. Uni, Z.; Ferket, P. R., 2004: Methods for early nutrition and their potential. World Poultry Science journal 60, 101–111. Zhai, W.; Gerard, P. D.; Pulikanti, R.; Peebles, E. D., 2011a: Effects of in ovo injection of carbohydrates on somatic characteristics and liver nutrient profiles of broiler embryos and hatchlings. Poultry Science 90, 2681–2688. Zhai, W.; Gerard, P. D.; Pulikanti, R.; Peebles, E. D., 2011b: Effects of in ovo injection of carbohydrates on embryonic metabolism, hatchability, and subsequent somatic characteristics of broiler hatchlings. Poultry Science 90, 2134– 2143. Zhai, W.; Rowe, D. E.; Peebles, E. D., 2011c: Effects of commercial in ovo injection of carbohydrates on broiler embryogenesis. Poultry Science 90, 1295–1301. Zhang, N.; Hartig, H.; Dzhagalov, I.; Draper, D.; He, Y., 2005: The role of apoptosis in the development and function of T lymphocytes. Cell Research 15, 749–769.
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ORIGINAL ARTICLE
In ovo carbohydrate supplementation modulates growth and immunity-related genes in broiler chickens S. K. Bhanja, A. Goel, N. Pandey, M. Mehra, S. Majumdar and A. B. Mandal Central Avian Research Institute, Izatnagar, UP, India
Summary A study was undertaken to investigate the role of in ovo administrated carbohydrates on the expression pattern of growth and immune-related genes. In ovo injections (n = 400) were carried out on the 14th day of incubation into the yolk sac/amnion of the broiler chicken embryos. Expression of growth-related genes: chicken growth hormone (cGH), insulin-like growth factor-I & II (IGF-I & II) and mucin were studied in hepatic and jejunum tissues of late-term embryo and early post-hatch chicks. Expression of candidate immune genes: Interleukin-2, 6, 10 and 12 (IL-2, IL-6, IL-10 and IL-12), Tumour necrosis factor-alpha (TNF-a) and Interferon gamma (IFN-c) were studied in peripheral blood monocyte cells of in ovo-injected and control birds following antigenic stimulation with sheep RBC (SRBC) or mitogen concanavalin A (Con-A). Glucose injection significantly increased the expression of IGF-II gene during embryonic period and both cGH and IGF-II in early post-hatch period, while ribose-injected chicks had higher expression of IGF-II gene during embryonic stage. Enhanced mucin gene expression was also observed in fructose-injected chicks during embryonic age. Glucose-injected chicks had higher expression of IL-6 or IL-10, while those injected with fructose or ribose had higher expression of IL-2, IL12 and IFN gamma. It is concluded that in ovo supplementation of carbohydrates might help in improving the growth of late-term embryos and chicks. In ovo glucose could modulate humoral-related immunity, while fructose or ribose might help in improving the cellular immunity in broiler chickens. Keywords in ovo injection, carbohydrates, gene expression, growth and immunity, broiler chickens Correspondence Dr. S. K. Bhanja, Principal Scientist, PHM Section, Central Avian Research Institute, Bareilly 243122, (Uttar Pradesh) India. Tel.: 91-9359105979; Fax: 91-5812301321; E-mail: [email protected] Received: 12 July 2013; accepted: 27 March 2014
During pre- and early post-hatch period, the chicks utilize their energy reserves to meet high demand for carbohydrates. As the amount of carbohydrates in the egg is very low, gluconeogenesis from protein is the source of glucose for accumulation of glycogen that eventually fuels the hatching activities (Klasing, 1998). When energy status is limited, hatchlings may lose weight and subsequently the development of critical tissues hampered. Moreover, in most of the commercial poultry operations, chicks get access to feed several hours after hatching. These limitations may be alleviated by administering refined carbohydrates or amino acids into the amnion or yolk sac at different days of incubation by a noble method called in ovo feeding, thus improving the chick’s liveability and growth (Bhanja et al., 2004b; Uni and Ferket, 2004). The in ovo fed nutrients help in accelerating enteric development in broilers and turkeys for greater
digestive and nutrient absorptive capacity during posthatch period (Uni and Ferket, 2004). It had already been reported that addition of glucose in drinking water suppressed gluconeogenic enzyme activity (Donaldson, 1995), thereby sparing the catabolism of useful proteins and antibodies. Studies conducted at our laboratory also revealed that glucose injection at later stage of embryonic development not only improved the broiler chick weight but also helped in the development of digestive tract (Bhanja et al., 2008a,b). Bhattacharyya et al. (2007) reported that in ovo injection of 1 ml, 10% glucose in turkey eggs on 25th day of incubation increased antisheep red blood cell (SRBC) titre (humoral immunity) and weight of bursa of fabricious at 28 days post-hatch, however, no affect was observed on cell-mediated immune response to PHAP (Phytohaemagglutinin-P). Earlier studies conducted on growth factors revealed that circulating IGF-1 and IGF-2 as well as hepatic IGF-1 mRNA expressions were regulated by
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Introduction
Carbohydrates and gene expressions
S. K. Bhanja et al.
nutritional state (Thissen et al., 1994). In avian species, expression of IGF-1 was regulated in a growth hormone (GH) dependent manner, during post-natal development (McMurtry et al., 1997). In spite of growth function, nutrients also play an important role in the functioning of the immune system. Upon activation, naive T helper cells (Th0) differentiate into either T helper 1 (Th1) or Th2 effector cells and secrete different cytokines that mediate immune responses (Spellberg and Edwards, 2001). Th1 type responses are primarily cell-mediated immunity and inflammation, while Th2 type are cytokines mediating humoral immunity. It will be interesting to see whether the nutrients could be able to modulate expression of certain cytokines, thus mediating humoral or cellular immunity. Scanty information is available on the role of critical macro/micro-nutrients on the expression of growth and immunity-related genes in chickens. Hence, keeping in view of the above facts, an effort was made to assess the effect of in ovo supplementation of carbohydrates (glucose, fructose or ribose) on the expression profile of growth and immune-related genes in broiler chickens during late embryonic and early post-hatch periods. Such understanding will help in formulating diets of neonatal chicks for better growth and immune responsiveness.
of the embryo, under laminar flow system, following standard procedure of Bhanja et al. (2004a). Briefly, first the target site (yolk sac/amnion) in the egg was standardized by injecting 0.5 ml solution of Indian ink through pinhole created at the broad end of a 14thday embryonated egg using different length of needles. Immediately after the injection, the shell of the respective end was cut with dental drill, and egg shell and shell membranes were removed with forceps. The injection site was visually observed. Based on the deposition site, the needle which reached to yolk sac/ amnion of 14th-day embryonated egg maximum time was selected for in ovo supplementation of nutrients. Before in ovo injection, the broad end of the egg was sterilized with 70% alcohol and a pin hole was made. With the help of syringe containing 24 gauge 25 mm hypodermic needle 0.5 ml sterile water containing carbohydrate (50 mg each) was injected into the yolk sac/amnion of embryo. After in ovo injection, the site was sealed with sterile paraffin and eggs were returned to the incubator. On the 19th day of incubation, the eggs were placed in pedigree-hatching boxes. Post-hatch, the chicks were reared in electrically operated battery brooders and provided standard nutrition and management in accordance with Institutional Ethics Committee at Central Avian Research Institute, Izatnagar, India till 42nd day of age.
Materials and methods
Collection of tissues for growth-related genes
Experimental birds and treatments
Four embryos/chicks from each group were killed at different ages (18th and 20th ED, day old, 7th, 10th and 14th day post-hatch). About 200 mg of liver (for cGH, IGF-I IGF-II gene expression) and intestinal tissue (jejunum, for mucin gene expression) from each bird were collected. The tissue was then homogenized using automated homogenizer (Polytron) to disrupt the cells and to remove the debris. 50 mg of homogenized tissue was used for total RNA isolation and the rest, 150 mg of tissue, was immersed in 500 ll RNA stabilizing solution and kept at 4 °C overnight and then transferred to 80 °C deep freeze for contingency uses.
The study was conducted using carbohydrates (glucose, fructose and ribose) as in ovo nutrients at a concentration of 50 mg/egg. 400 fertile eggs collected from coloured synthetic broiler breeder flocks (Cross of Cornish and Plymouth Rock) maintained on balanced ration were distributed into four groups (threetreatments and one un-injected control) of 100 eggs each. In our earlier studies on in ovo feeding (Bakyaraj et al., 2012), no statistical difference was observed with respect to percent hatchability and post-hatch performance of un-injected control group and sham control (only placebo no nutrients) group; hence, sham control group was excluded from the present experimental design. After collection, the eggs were fumigated (at 19 concentration) immediately and incubated in a forced draft incubator at a temperature of 37.5 °C with relative humidity of 60%.
Differential expression of candidate immune function genes
In ovo administration of nutrients were carried out on the 14th day of incubation into the yolk sac/amnion
To quantify the candidate genes of humoral immunity like IL-6, IL-10 and TNF-a, four birds from each group were injected intravenously with 1.0 ml, 1% (V/V) sheep red blood cells (SRBC) on 14th day post-hatch and 5 days post-injection, blood was collected from jugular vein. Approximately 1.5 ml blood from each
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bird was mixed with 3.0 ml of PBS (1:2). For separation of peripheral blood mononuclear cells (PBMC), 3.0 ml Histopaque-1.077 (Sigma Diagnostics, St. Louis, MO, USA) was taken in a 15.0 ml conical centrifuge tube, and the diluted blood was carefully layered over the histopaque and centrifuged at 471 g for exactly 20 min at room temperature. After centrifugation, the opaque interface containing PBMC was carefully transferred to a clean conical centrifuge tube and 10.0 ml isotonic PBS solution was added and mixed by gentle aspiration then centrifuged at 910 g for 10 min. This step was repeated twice to ensure complete washing. After centrifugation, pellet was dissolved in denaturing solution for total RNA isolation. For quantification of cellular immune-related genes like IL-2, IL-12 and IFN gamma, approximately, 1.5 ml blood was collected from the jugular vein of four birds per group on the 21st day post-hatch and PBMC cells were separated (as detailed earlier). Viability of these cells were determined by Trypan blue (0.4%) staining method. Cells were re-suspended in known volume of RPMI-1640 (Sigma) to make the final concentration of 106 cells per ml of medium. PBMCs were then plated in 6-well culture plates with RPMI-1640 (without phenol red) medium, supplemented with 10% FBS, 2 mM L-glutamine, 2 mM L-argenine under 5% CO2 tension in humidified atmosphere. The PBMC cells were sensitized to 10 lg/ml concanavalin-A (Con-A) for a period of 4 h; then the PBMCs adhered to culture plates were harvested in to a 1.5 ml eppendorf tube and centrifuged at 3640 g for 1 min. After centrifugation, the pellet was dissolved in denaturing solution for total RNA isolation.
Carbohydrates and gene expressions
First strand cDNA synthesis by RT-PCR
Concentration of each RNA sample was computed and then adjusted with RNA free water to make a uniform concentration (2000 ng/ll). Required amount of RNA from each sample was taken as template and first strand cDNA was prepared by Revert AidTM first strand cDNA synthesis kit (MBI, Fermentas Inc., Hanover, MD, USA). This first stand cDNA was used as template for amplification of different genes by polymerase chain reaction (PCR) or stored at ultra-low temperature for further use. Standardization of primers for PCR
Oligonucleotide primers (Table 1) were designed, synthesized and standardized for PCR reaction conditions. PCR products of different genes were sequenced for nucleotide bases and the sequences were submitted to NCBI, USA or EMBL, UK for confirmation, and accession numbers were obtained for those genes . Polymerase chain reaction
The PCR amplification was carried out in 25 ll volume, containing 10 pM of each primer, 0.1 mM dNTP mix, 1 unit of Taq DNA polymerase (Fermentas) and 1 ll cDNA in 19 Taq polymerase buffer. Cycling conditions were: initial denaturation at 94 °C (5 min); 35 cycles of denaturation at 94 °C (45s), annealing temperature (as given in Table 1) for 1 min and extension at 72 °C (1 min); and final extension at 72 °C (10 min). Semi-quantitative analysis
Total RNA isolation
The samples collected for expression of growth and immunity-related genes were subjected to total RNA isolation (RNAgents, Promegaâ, Madison, WI, USA) as per the manufacture instructions. The concentrations and purities of RNA preparations were determined spectrophotometrically by taking absorbance at 260 and 280 nm in a Nanodrop instrument. It was ensured that A260/A280 ratio of the samples was >1.8. The integrity of RNA was also assessed by UV visualization of intact 28S and 18S bands on a 2% agarose gel. The possible trace of genomic DNA was removed by treating 5 lg of RNA samples with 5 U of RNase-free DNase at 37 °C for 1 h. The DNase was subsequently inactivated by incubating at 65 °C for 10 min. The RNA samples were stored at –80 °C till further processing. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
Electrophoresis was performed on 2% Agarose having Ethedium Bromide stain at 2 V/cm for 2 to 3 h. The gel was then examined under UV light and photographed in a Gel documentation system (Bio-Rad). Image J analysis software was used for quantification of luminance (integrated density). Housekeeping (28S rRNA) gene expression of respective sample was used for normalization of target gene expression. Ratio of gene expression for each sample was calculated using the formula: Ratio expression integrated density of target gene ¼ Integrated density of housekeeping gene (28S rRNA) The average ratio of un-injected control group was used to calculate the fold change in target 3
4
Interleukin-6 (IL-6)
Interleukin-10 (IL-10)
Tumour Necrosis Factor –alfa (TNFa)
Interleukin-2 (IL-2)
Interleukin-12 (IL-12)
5
6
7
8
9
28S r RNA (Reference)
Mucin gene
4
11
Insulin-like growth factor-II (IGF-II)
3
Interferon gamma (IFNc)
Insulin-like growth factor-I (IGF-I)
2
10
Chicken Growth hormone gene (cGH)
Gene
1
Sl No. F-caccacagctagagacccacatc R-cccaccggctcaaactgc F-ggtgctgagctggttgatgc R-cgtacagagcgtgcagatttaggt F-ggcggcaggcaccatca R-cccggcagcaaaaagttcaag F-ctggctccttgtggctcctc R-agctgcatgactggagacaactg F-gaaatccctcctcgccaatctga R-tgaaacggaacaacactgccatct F-ctgaaggcgacgatgc R-ttcctcctcctcatcagc F-agaccagatgggaagggaatgaa R-gaagaggccaccacacgacag F-cccgtggctaactaatctgctg R-tgagacaccagtgggaaacagt F-gccgactgagatgttcctgg R-ccttgcttttgtatttctttgtgc F-agctgacggtggacctattattgt R-cggctttgcgctggattc F-caggtgcagatcttggtggtagta R- gctcccgctggcttctcc
Primer (5I–3I)
Table 1 Oligonucleotide sequence of growth and immune-related gene primers
58
58
57
57
55
57
55
58
58
58
58
Annealing Temp. (oC)
273
260
227
287
219
179
219
242
215
203
201
Size of Amplicon (bp)
House keeping
Cell mediate immunity
Cell mediate immunity
Cell mediate immunity
Humoral Immunity
Humoral Immunity
Humoral Immunity
Intestinal tract Dev.
Growth
Growth
Growth
Putative biological role
JN639848
JN942588
JN942590
HE608819
JN942589
EF554720
JN639847
JN639849
JN942579
JN942578
HE608816
Accession number
Carbohydrates and gene expressions S. K. Bhanja et al.
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Carbohydrates and gene expressions
gene expression of treated samples using the formula: Fold Change (each sample) Ratio (each sample) ¼ Average ratio of un-injected control group The resulted fold change in gene expression of each treatment group was used for statistical analysis. As the average ratio of un-injected control group was used to find the fold change in the treatment groups, so the fold change in un-injected control group was always one.
afterwards, 100 lg PHA-P, dissolved in 0.1 ml PBS, was injected into the same interdigital space of the right foot. The toe web of the left foot was used as control and injected only with PBS. The inflammatory response was determined 24 h later by measuring the thickness of the respective toe-webs and subtracting the earlier measurements by using formula (Corrier and Deloach, 1990), Foot Web Index = (R2–R1)–(L2– L1) where R2 – thickness after 24 h of PHA-P injection, R1 – thickness before injection of PHA-P injection, L2 – thickness after 24 h of PBS injection and L1 – thickness before injection of PBS injection. Statistical analysis
In-vivo immune responses
Sheep red blood cell suspended in Alsever’s solution were washed three times in isotonic phosphatebuffered saline (PBS: pH 7.2) using centrifugation (328 g) and adjusted to provide a 1% suspension (v/v) which was stored at 4 °C prior to use. Ten chicks from each group (3-week-old) were injected intravenously with 1 ml of the SRBC suspension. Five days later, a blood sample (2.0 ml) was obtained from the jugular vein of each chick. Each blood sample was allowed to clot for serum collection and sera were stored at 20 °C until analysis. The antibody response to SRBC was determined using a standard haemagglutination assay (Siegel and Gross, 1980). The reciprocal of highest dilution showing clear agglutination was the end point of titre and the values were expressed as log 2. The cellular immune response was also assessed in another ten chicks (3-week-old) using the in-vivo cutaneous basophilic hypersensitivity response to the lectin phytohaemagglutinin from Phaseolus vulgaris (PHA-P). The toe web thicknesses, between the third and fourth digits, of both left and right feet were measured using a micrometre. Immediately
Fold changes in expression of growth-related genes for a particular age was analysed by one-way ANOVA, whereas the multivariate analysis (taking treatment and age as factors) was done to see treatment/period effect using SPSS software package Ver 16.0 (2007). Difference in mean values was considered as significant at the level of 95% (p < 0.05) and 99% (p < 0.01) using Duncan’s multiple range test (Duncan, 1955). The growth response data and immunological parameters were analysed by one-way ANOVA only. Results Expression of growth-related genes
Gel photographs showing integrated densities of target gene and a housekeeping gene (28s ribosomal RNA) for a particular sample during pre- and post-hatch period have been presented in Figures 1 and 2. Variation in the intensities of target and housekeeping gene was noticed within and between groups; the ratio of relative intensity was calculated to derive the fold expression as described in the earlier section and are presented in Table 2 and Figure 3.
(a)
(b) (c)
Fig. 1 Gel photographs showing band intensity (four per treatment) of growth-related genes and a house-keeping gene (28s RNA) during embryonic stages in in ovo glucose- (G), fructose- (F) or ribose (R)-injected and un-injected control (C) embryos. (a) Glucose showing higher hepatic cGH and ribose showing higher IGF-II gene expression on 18th day of embryonic age (b) Fructose showing higher jejunal mucin gene expression on 18th day of embryonic age (c) Fructose and ribose showing higher hepatic cGH gene expression on 20th day of embryonic age.
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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Carbohydrates and gene expressions
(a)
(b) (c) (d) Fig. 2 Gel photographs showing band intensity (four per treatment) of growth-related genes and a house-keeping gene (28s RNA) during post-hatch periods in in ovo glucose- (G), fructose- (F) or ribose (R)-injected and un-injected control (C) chicks. (a) Glucose showing higher hepatic cGH, IGF-I and IGF-II gene expressions on 7th day post-hatch age (b) Glucose showing higher hepatic cGH gene expression on 10th day post-hatch age (c) Glucose showing higher hepatic IGF-I gene expressions on 14th-day post-hatch age (d) Ribose showing higher jejunal mucin gene expressions on 7th-day posthatch age.
Pre-hatch period
When data was analysed by two way ANOVA to see period and treatment interaction, irrespective of period of study higher hepatic cGH (1.43–1.58-fold) and IGF-I (1.15–1.28) expression but not differing (p > 0.05) from control was observed in all the carbohydrate-injected embryos. Hepatic cGH expression was apparently higher in glucose (18th ED) or in fructose or ribose-injected embryos (20th ED) than un-injected embryos (Figure 3). Whereas IGF-II expression was higher (p < 0.05) in glucose and ribose-injected embryos compared with control embryos, the fructose group had intermediate values. Higher mucin gene expression (p < 0.01) was observed in fructose-injected embryos. When expression was compared for different embryonic ages,
higher hepatic cGH at 18th or 20th ED, IGF-II and mucin gene expression at 18th ED was observed than those at DOH (Table 2). There was no significant interaction between period and treatment in cGH or IGF-I expression. However, interaction was significant for IGF-II and mucin gene where glucoseor ribose-injected embryos had higher IGF-II expression and fructose-injected embryos had higher mucin gene expression at 18th ED (Figure 3). Post-hatch period
When expression was compared for different posthatch (PH) periods cGH and mucin gene expression was higher on 7th and 10th PH, while IGF-I and IGFII expression was higher only on 7th PH. Between treatments cGH and IGF-II gene expression was
Table 2 Mean fold change in the expression pattern of growth-related genes in hepatic and jejunal tissues of four embryos/chicks in different carbohydrate treatments at pre- and post-hatch period Embryonic
Age 18th ED 20th ED DOH Treatments Glucose Fructose Ribose Control SEm Significance (Probability) Period Treatment Period 9 Treatment
Post-hatch
cGH
IGF-I
IGF-II
Mucin
1.46b 1.71b 0.95a
1.29 1.03 1.12
3.05b 1.16a 0.94a
1.43b ND 1.04a
1.43 1.49 1.58 1.00 0.107
1.16 1.28 1.15 1.00 0.061
2.11b 1.64ab 2.12b 1.00a 0.234
1.07a 1.71b 1.16a 1.00a 0.097
0.01 NS NS
NS NS NS
0.01 0.05 0.01
0.01 0.01 0.01
cGH
IGF-I
IGF-II
Mucin
7th PH 10th PH 14th PH
1.67c 1.27b 0.95a
1.38b 0.99a 0.90a
1.23b 1.03a 1.11ab
1.22b 1.01b 0.68a
Glucose Fructose Ribose Control SEm
1.66b 1.22a 1.31a 1.00a 0.081
1.22 1.18 0.97 1.00 0.062
1.36b 1.12a 1.01a 1.00a 0.038
0.73a 0.94ab 1.21b 1.00ab 0.063
0.01 0.01 0.01
0.01 NS 0.01
0.01 0.01 0.05
0.01 0.01 NS
Values are mean of four observations. Means bearing different superscripts in a column differ significantly (p < 0.05).
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Carbohydrates and gene expressions
(a)
(b)
(c)
(d)
Fig. 3 Mean fold increase in the expression pattern of growth-related genes during embryonic and post-hatch period in glucose-, fructose- or riboseinjected embryos compared with un-injected control embryos. (a) Hepatic chicken growth hormone (b) Hepatic insulin-like growth factor-I (c) Hepatic insulin-like growth factor-II (d) Jejunal mucin gene.
significantly higher in glucose-injected chicks. No variation was observed in the expression of IGF-I gene; however, the expression of mucin gene was significantly higher in ribose-injected chicks than glucoseinjected chicks (Table 2). Significant interaction between period and treatment was observed for cGH, IGF-I and IGF-II gene, where in ovo glucose-injected chicks had higher expression of hepatic cGH on 7th and 10th PH, IGF-I gene on 7th PH and IGF-II expression on 7th and 14th day PH than control chicks (Figure 3). Expression of immune-related genes
The relative expression of humoral immunityrelated genes (IL-6, IL-10 and TNF alpha) have been presented in Figure 5. Glucose- or fructoseinjected chicks had significantly higher expression (p < 0.01) of IL-6 gene compared with control, but down-regulated expression was observed in ribosetreated birds. Expression of IL-10 was higher (p < 0.01) in glucose-injected chicks, but the expression decreased in fructose- or ribose-injected chick compared to control. There was no variation in the expression of TNF alpha gene between glucose or fructose treatment and control, but, ribosetreated chicks showed significant down-regulation (p < 0.01) of TNF alpha gene. The relative expression of cellular immunity cytokines, IL-2, IL-12 and IFN gamma at 14th day
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
post-hatch have been presented in Figure 4 and 5. Expression of IL-2 gene was significantly higher (p < 0.01) in fructose- or ribose-injected chicks compared with un-injected control; however, the expression in glucose-injected chicks was down-regulated in comparison to fructose- or ribose-injected chicks. Expression of IL-12 gene was significantly higher (p < 0.05) in ribose-treated chicks than those of glucose, but no difference was observed between carbohydrate-treated chicks and control. Expression of IFN gamma was significantly (p < 0.01) higher in ribose- or fructose-injected birds compared with control, but down-regulated in glucose-injected chicks. In-vivo immune response to SRBC and PHA-P
Humoral immunity (response to SRBC antigen measured as HA titre, Log2 value) was higher (p < 0.05) in ribose-injected chicks than control; however, titre
Fig. 4 Gel photographs showing band intensity of Cell-Mediated Immune genes and a house-keeping gene (28s RNA) at 21 d post-hatch in in ovo glucose- (G), fructose- (F) or ribose (R)-injected and un-injected control (C) chicks after sensitizing the PBMC cells with 10 lg Con-A/mL of RPMI-1640 media.
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Carbohydrates and gene expressions
(a)
(b)
Fig. 5 Differential expression of immunity-related genes showing mean fold change over control group in in ovo carbohydrates-injected chicks (a) Glucose- or fructose-treated chicks showing higher expression of humoral immune-related genes in PBMC following SRBC challenge (b) Fructoseor ribose-treated chicks showing higher expression of cellular immune genes in PBMC after sensitizing with Con-A.
Table 3 Effect of in ovo injection of carbohydrates on the antibody titre (response to SRBC injection) and toe web thickness (response to PHAP) of ten 3-week-old broiler chickens
In the present study, in ovo glucose injection resulted in significantly higher expression of cGH gene during post-hatch period. Bhanja et al. (2008b) reported that in ovo injection of glucose on 14th day of incubation did not affect the hatchability and chick weight but had higher levels of plasma glucose and plasma protein on the DOH. In a recent study, Zhai et al. (2011a, b,c) reported that embryo weight relative to set egg weight on 19th day of incubation increased by injection of carbohydrates (glucose, sucrose, maltose and dextrin). These findings corroborate our result, as cGH
expression was on peak at 20th day of incubation. Moreover, studies conducted at our laboratory on in ovo feeding of amino acids and vitamins had significantly higher cGH expression (Bhanja, S.K., personal communication), Thus suggesting the role of in ovo feeding of nutrients for enhancing overall growth and proliferation of tissues in neonatal chicks. IGF-I levels increased steadily during incubation from day 6 to peak at day 15 of incubation but reduced during hatch (Robcis et al., 1991). In the present study also, the expression of hepatic IGF-1 gene was apparently higher on the 18th day of incubation and showed a decreasing trend towards hatch. During post-hatch period, higher IGF-1 expression was observed on 7th than 10th or 14th PH in glucose-injected chicks. Contrary to this Lu et al. (2007) reported that after hatch, IGF-1 levels rapidly increased and kept increasing all the way until 21 days post-hatch. Deprem and G€ ulmez (2007) reported that in ovo rhIGF-1 injection in quails was found to accelerate skeletal muscle development in quail embryos. The expression of cGH gene was higher at 18th and 20th ED but no variation was observed in the expression of IGF-I gene during this period suggesting IGF-I gene expression is GHindependent during embryonic stage as that of reported earlier (Tanaka et al., 1996) but acts as a mediator of many of the actions of GH and stimulator of tissue growth and differentiation in post-hatch period (Cohick and Clemmons, 1993). In this study also, during post-hatch period the expression of cGH and IGF-1 gene followed a similar trend and in ovo glucose injection had higher expression of both the genes although not significant for IGF-I. Foye (2005) reported that in poultry as in mammalian species, IGF-I and II stimulate hepatic glycogen, RNA and protein synthesis. The expression of IGF-II gene during incubation was found to be highest at 18th ED; however, during PH, the expression had
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Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
Treatments
Anti-SRBC Titre (log 2)
Foot web Index (mm) in response to mitogen PHAP
Glucose Fructose Ribose Control Pooled SEM Sig. Level
9.71ab 9.71ab 10.83b 9.00a 0.22 p = 0.022
0.3500a 0.4650ab 0.6575b 0.4729ab 0.040 p = 0.039
Values are mean of ten observations. Means bearing different superscripts in a column differ significantly (p < 0.05).
value in glucose or fructose-injected chicks were not different from ribose or control chicks (Table 3). Cellular immunity (foot web index, response to PHA-P injection) was similar among carbohydrate-injected and un-injected control chicks, although response was better in ribose-injected chicks (Table 3) but it did not reach to a significant level (p > 0.05). Discussion Growth-related genes
S. K. Bhanja et al.
shown decreasing trend with increase in age. This correlates the earlier findings where IGF-II is reported as an important functionary for chick embryonic development (McMurtry et al., 1998) and plasma IGF-II level decreased from 60 ng/ml on 17th of incubation to 25 ng/ml at 21 day post-hatch (Lu et al., 2007). Fructose-injected embryos had higher expression of jejunal mucin gene at 18 ED, whereas, ribose-injected chick had significantly higher expression on 7th day post-hatch than glucose-injected chicks. Uni and Ferket (2004), reported that in ovo feeding of carbohydrates at 18 day of incubation resulted significant increase in jejunum villi height by over 45% even 48 h after injection. Higher mucin gene expression and goblet cell density in jejunum villi was reported in in ovo carbohydrate-injected embryos/chicks than the saline-control group (Smirnov et al., 2006). Bhanja et al. (2008a) had also reported better gastrointestinal tract development in glucose-injected chicks. In another study, in ovo mannan oligosaccharide feeding at 17th or 18th day of incubation, resulted in the 3 -fold increase of mucin 2 gene expression (CheledShoval et al., 2011). These studies suggested that the presence of carbohydrate in the intestinal lumen of the chicken embryo might have triggered the goblet cells for production of acidic mucin. From our study, the role of fructose in enteric development cannot be ruled out as it increased the mucin gene expression during pre-hatch period.
Carbohydrates and gene expressions
Development of both B and T lymphocytes is initiated during embryogenesis and continues till post-hatch. These cells play an important role in immunity as native T cells differentiate into Th1 cells (cellular immunity) and Th2 cells (humoral immunity). Glick et al. (1981, 1983), reported that the thymus is very sensitive to periods of food deprivation, which caused a rapid decline in CD4+ T cells resulting in lower IgG production. The CD4+ cells act as the source for IL-6, a pro-inflammatory cytokines, which induces the final maturation of B cells into antibody-secreting plasma cells (Jones, 2005) causing the proliferation and differentiation of immunoglobins. In the present study, expression of humoral immune-related genes, that is, IL-6 and IL-10, were increased in glucose treatment group in comparison to un-injected control. This is in line with the earlier study of Greiner et al. (1994), who reported that glucose is an essential fuel for proliferating Th2 cells for the production of antibody, thus providing humoral immunity. Studies in human
diabetic individuals revealed that hyperglycaemia acutely increased circulating cytokine concentrations of IL-6 and TNF-a by an oxidative mechanism and played a role in the immune activation in diabetes (Esposito et al., 2002). In another study, Humphrey and Rudrappa (2008) reported that glucose availability induced metabolic changes in thymocytes that altered their energy status and influence the development and differentiation of T cells in the chicken thymus (Zhang et al., 2005). They had also reported that thymocytes developing in low glucose concentrations (5 mM) had elevated rates of apoptosis. Therefore, increasing the in ovo availability of glucose might have increased the synthesis of trophic factors which promote T-cell glucose metabolism and helps in the development of the chick’s immune system at early life. In-vivo response to SRBC antigen was significantly higher in ribose-injected birds than un-injected control but not different from that of glucose- or fructoseinjected chicks. However, Bhattacharyya et al. (2007) reported that in ovo injection of 1 ml of 10% glucose to turkey eggs increased anti-SRBC titre (humoral immunity) and weight of bursa of fabricious. It is reported that Th1 and Th2 responses are also cross-regulated, wherein IFN-gamma, a cytokine produced by Th1 cells, inhibits IL-4 production and suppresses Th2 development (Gajewski and Fitch, 1988). Conversely, IL-4 and IL-10 produced by Th2 cells block differentiation of Th0 to Th1 (D’Andrea et al., 1993). In the present study, expression of IL-10 was higher in glucose-injected chicks, but the expression decreased in fructose or ribose-injected chick compared to control chicks. It was also interesting to note that the expression of IL-2 and IFN gamma gene was significantly higher in fructose or ribose treated chicks but no difference was observed in glucose treatment as compared to control. Earlier studies on critical amino acids had demonstrated that either in ovo feeding (Bhanja et al., 2012) or dietary supplementation (Sijben et al., 2001) increases the PHA-P response and expression of cellular immune genes (IL-2 and IL-12) in broiler chicks. Similarly, Bhanja and Mandal (2005) also found a significant difference in cell-mediated immunity in in ovo injection of amino acid. The present study on cellular immune response to mitogen PHA-P revealed no significant difference between carbohydrates-injected and control chicks, but apparently higher response was observed in ribose-injected chicks. In another study, Kadam et al. (2008) reported that in ovo injection of threonine in broiler eggs had no affect on cell-mediated immune response to PHA-P.
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Immunity-related genes
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Carbohydrates and gene expressions
In summary, the present investigation on in ovo feeding of carbohydrates revealed that glucose played an important role during late embryonic and early post-hatch period by increasing the expression of cGH and IGF-II genes. It also increased expression of humoral genes, IL-6 and IL-10. The role of in ovo supplemented fructose and ribose could not be ignored as their presence during late embryonic stage resulted in better expression of IGF-II or mucin gene. Additionally, ribose modulated the expression of cellular References Bakyaraj, S.; Bhanja, S. K.; Majumdar, S.; Dash, B. B., 2012: Post-hatch immunomodulation through in ovo supplemented nutrients in broiler chickens. Journal of the Science of Food and Agriculture 92, 313–320. Bhanja, S. K.; Mandal, A. B., 2005: Effect of in ovo injection of critical amino acids on pre and post hatch growth, immunocompetence and development of digestive organs in broiler chickens. AsianAustralasian Journal of Animal Science 18, 524–531. Bhanja, S. K.; Mandal, A. B.; Johri, T. S., 2004a: Standardization of injection site, needle length, embryonic age and concentration of amino acids for in ovo injection in broiler breeder eggs. Indian Journal of Poultry Science 39, 105–111. Bhanja, S. K.; Mandal, A. B.; Goswami, T. K., 2004b: Effect of in ovo injection of amino acids on growth, immune response, development of digestive organs and carcass yield of broiler. Indian Journal of Poultry Science 39, 212– 218. Bhanja, S. K.; Mandal, A. B.; Agarwal, S. K.; Majumdar, S.; Bhattacharyya, A.; Kadam, M., 2008a: In ovo glucose injection for higher chick weight and gastrointestinal tract development. The Indian Veterinary Journal 85, 289–292. Bhanja, S. K.; Mandal, A. B.; Agarwal, S. K.; Majumdar, S., 2008b: Effect of in ovo glucose injection on the post hatchgrowth, digestive organ development and blood biochemical profiles in broiler chickens. Indian Journal of Animal Science 78, 869–872. Bhanja, S. K.; Mandal, A.; Agarwal, S. K.; Majumdar, S., 2012: Modulation of post hatch-growth and immunocompetence through in ovo injection of limiting amino acids in broiler chickens. Indian Journal of Animal Science 92, 993–998.
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immune genes, IL-2, IFN gamma and IL-12 in broiler chickens. Acknowledgement The financial assistance provided by Department of Biotechnology, Ministry of Science and Technology, Govt. of India for carrying out this research work is duly acknowledged.
Bhattacharyya, A.; Majumdar, S.; Bhanja, S. K.; Mandal, A. B.; Dash, B. B.; Agarwal, S. K., 2007: In ovo injection of glucose supports immunecompetence. World Poultry–Turkey Special 10-11. Cheled-Shoval, S. L.; Amit-Romach, E.; Barbakov, M.; Uni, Z., 2011: The effect of in ovo administration of mannan oligosaccharide on small intestine development during the pre- and posthatch periods in chickens. Poultry Science 90, 2301–2310. Cohick, W. S.; Clemmons, D. R., 1993: The insulin-like growth factors. Annual Review of Physiology 55, 131–153. Corrier, D. E.; Deloach, J. R., 1990: Evaluation of cell mediated, cutaneous basophil hypersensitivity in young chickens by inter digital skin test. Poultry Science 69, 403–408. D’Andrea, A.; Aste-Amezaga, M.; Valiante, N. M.; Ma, X.; Kubin, M.; Trinchieri, G., 1993: Interleukin 10 (IL-10) inhibits human lymphocyte interferon-gamma production by suppressing natural killer cell stimulatory factor/ IL-12 synthesis in accessory cells. Journal of Experimental Medicine 178, 1041–1048. Deprem, T.; G€ ulmez, N., 2007: The effects of in ovo insulin-like growth factor-1 on embryonic development of musculus longus colli dorsalis in japanese quail. Turkish Journal of Veterinary and Animal Science 31, 233–240. Donaldson, W. E., 1995: Carbohydrate, hatchery stressors affect poult survival. Feedstuffs 67, 16–17. Duncan, D. B., 1955: Multiple range and multiple F tests. Biometrics 11, 1–42. Esposito, K. F.; Nappo, R.; Marfella, G.; Giugliano, F.; Giugliano, M. C.; Quagliaro, L.; Ceriello, A.; Giugliano, D., 2002: Inflammatory cytokine concentrations are acutely increased by hyperglycemia in humans: role of oxidative stress. Circulation 106, 2067–2072.
Foye, O. T., 2005: ‘The biochemical and molecular effects of amnionic nutrient administration, “in ovo feeding” on intestinal development and function and carbohydrate metabolism in turkey embryos and poults’, Dissertation Ph.D, North Carolina State University, Raleigh, NC. Gajewski, T. F.; Fitch, F. W., 1988: Antiproliferative effect of IFN gamma in immune regulation. I. IFN-gamma inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones. Journal of Immunology 140, 4245–4252. Glick, B.; Day, E. J.; Thompson, D., 1981: Calorie-protein deficiencies and the immune response of the chicken I. Humoral immunity. Poultry Science 60, 2494–2500. Glick, B.; Taylor, R. L. Jr; Martin, D. E.; Watabe, M.; Day, E. J.; Thompson, D., 1983: Calorie-protein deficiencies and the immune response of the chicken II. Cell-mediated immunity. Poultry Science 62, 1889–1893. Greiner, E. F.; Guppy, M.; Brand, K., 1994: Glucose is essential for proliferation and the glycolytic enzyme induction that provokes a transition to glycolytic energy production. Journal of Biological Chemistry 269, 31484–31490. Humphrey, B. D.; Rudrappa, S. G., 2008: Increased glucose availability activates chicken thymocyte metabolism and survival. Journal of Nutrition 138, 1153–1157. Jones, S. A., 2005: Directing transition from innate to acquired immunity: defining a role for IL-6. Journal of Immunology 175, 3463–3468. Kadam, M. M.; Bhanja, S. K.; Mandal, A. B.; Thakur, R.; Vasan, P.; Bhattacharyya, A.; Tyagi, J. S., 2008: Role of in ovo threonine supplementation on early growth, immunocompetence and digestive enzyme activity in broiler chickens. British Poultry Science 49, 736–741.
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S. K. Bhanja et al.
Klasing, K. C., 1998: Carbohydrates. In Comparative Avian Nutrition. CAB Intlernational, New York, pp. 201–209. Lu, J. W.; McMurtry, J. P.; Coon, C. N., 2007: Developmental changes of plasma insulin, glucagon, insulin-like growth factors, thyroid hormones, and glucose concentrations in chick embryos and hatched chicks. Poultry Science 86, 673– 683. McMurtry, J. P.; Francis, G. L.; Upton, Z., 1997: Insulin-like growth factors in poultry. Domestic Animal Endocrinology 14, 199–229. McMurtry, J. P.; Rosebrough, R. W.; Brocht, D. M.; Francis, G. L.; Upton, Z.; Phelps, P., 1998: Assessment of developmental changes in chicken and turkey insulin-like growth factor-II by homologous radioimmunoassay. Journal of Endocrinology 157, 463–473. Robcis, H. L.; Caldes, T.; De Pablo, F., 1991: Insulin-like growth factor-I serum levels show a midembryogenesis peak in chicken that is absent in growthretarded embryos cultured ex ovo. Endocrinology 128, 1895–1901. Siegel, P. B.; Gross, W. B., 1980: Production and persistency of antibodies in chickens to sheep erythrocytes
Carbohydrates and gene expressions
I. Directional selection. Poultry Science 59, 1–5. Sijben, J. W. C.; Schrama, J. W.; Parmentier, H. K.; Van der Poel, J. J.; Klasing, K. C., 2001: Effects of dietary polyunsaturated fatty acids on in vivo splenic cytokine mRNA expression in layer chicks immunized with salmonella typhimurium lipopolysaccharide. Poultry Science 80, 1164–1170. Smirnov, A.; Tako, E.; Ferket, P. R.; Uni, Z., 2006: Mucin gene expression and mucin content in the chicken intestinal goblet cells are affected by in ovo feeding of carbohydrates. Poultry Science 85, 669– 673. Spellberg, B.; Edwards, J. E. Jr, 2001: Type 1/Type 2 immunity in infectious diseases. Clinical Infectious Diseases 32, 76– 102. SPSS 2007: SPSS for Windows, Version 16.0. Released 2007. SPSS, Chicago. Tanaka, M.; Hayashida, Y.; Sakaguchi, K.; Ohkubo, T.; Wakita, M.; Hoshino, S.; Nakashima, K., 1996: Growth hormone independent expression of insulin-like growth factor I. messenger ribonucleic acid in extra hepatic tissues of the chicken. Endocrinology 137, 30–34.
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Thissen, J. P.; Ketelslegers, J. M.; Underwood, L. E., 1994: Nutritional regulation of insulin-like growth factors. Endocrinology Review 15, 80–101. Uni, Z.; Ferket, P. R., 2004: Methods for early nutrition and their potential. World Poultry Science journal 60, 101–111. Zhai, W.; Gerard, P. D.; Pulikanti, R.; Peebles, E. D., 2011a: Effects of in ovo injection of carbohydrates on somatic characteristics and liver nutrient profiles of broiler embryos and hatchlings. Poultry Science 90, 2681–2688. Zhai, W.; Gerard, P. D.; Pulikanti, R.; Peebles, E. D., 2011b: Effects of in ovo injection of carbohydrates on embryonic metabolism, hatchability, and subsequent somatic characteristics of broiler hatchlings. Poultry Science 90, 2134– 2143. Zhai, W.; Rowe, D. E.; Peebles, E. D., 2011c: Effects of commercial in ovo injection of carbohydrates on broiler embryogenesis. Poultry Science 90, 1295–1301. Zhang, N.; Hartig, H.; Dzhagalov, I.; Draper, D.; He, Y., 2005: The role of apoptosis in the development and function of T lymphocytes. Cell Research 15, 749–769.
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